Affinity peptides

ABSTRACT

Fusion proteins and a process for their purification by means of metal chelate affinity chromatography on NTA resins are provided by this invention.

This is a division of application Ser. No. 07/158,962 filed Feb. 22,1988, now allowed.

TECHNICAL FIELD

This invention relates to fusion proteins containing affinity peptides,genes coding for such proteins, expression vectors and transformedmicroorganisms containing such genes, and methods for the purificationof the fusion proteins.

BACKGROUND OF THE INVENTION

The possibility of preparing hybrid genes by gene technology has openedup new routes for the workup of recombinant proteins. By linking thecoding gene sequence of a desired protein to the coding gene sequence ofa protein fragment having a high affinity for a ligand (affinitypeptide), it is possible to purify desired recombinant proteins in theform of fusion proteins in one step using the affinity peptide.

By site-directed mutagenesis it is also possible to introduce specificchemical or enzymatic cleavage sites at the point of linkage of theaffinity peptide and the desired recombinant protein, so that after thepurification of the fusion protein by means of a suitable affinityresin, the desired recombinant protein can be recovered by chemical orenzymatic cleavage. Such purification methods have been described, forexample, in Science 198, 1056-1063 (1977) (Itakura et al.); Proc. Natl.Acad. Sci. U.S.A. 80, 6848-6852 (1983) (Germino et al.); Nucleic AcidsRes. 13, 1151-1162 (1985) (Nilsson et al.): Gene 32, 321-327 (1984)(Smith et al.). and European Patent Applications publication Nos. 150126 and 184 355.

SUMMARY OF THE INVENTION

This invention provides affinity peptides having at least twoneighbouring histidine residues which are especially suitable for thepurification of recombinant proteins by means of metal chelate affinitychromatography in nitrilotriacetic acid (NTA) resins. These affinitypeptides can be distinguished from the previously known peptidesprimarily in that they permit the problem-free purification of nativeand denatured proteins by means of NTA resins.

More particularly, this invention provides fusion proteins which containone or two affinity peptides, which peptides contain neighbouringhistidine residues, and a biologically active polypeptide or proteinlinked directly or indirectly to this/these affinity peptide(s); aprocess for their preparation by means of recombinant DNA technology anda process for their purification by means of metal chelate affinitychromatography in NTA resins. The present invention also provides geneswhich code for these fusion proteins, expression vectors which containthese genes, microorganisms transformed with these expression vectorsand processes for the preparation of said genes, expression vectors andtransformed microorganisms.

The affinity peptides of the fusion proteins of the invention aredefined by the general formula

    R.sup.1 --(His).sub.2-6 --R.sup.2

wherein R¹ represents hydrogen or from 1 to about 30 amino acids; and R²represents Q, Q-Ile-Glu-Gly-Arg- or Q-Asp-Asp-Asp-Asp-Lys-, where Q is apeptide bond or from 1 to about 30 amino acids. Especially preferredaffinity peptides have the following amino acid sequences:

Met-His-His,

Met-His-His-His,

Met-His-His-His-His,

Met-His-His-His-His-His,

Met-His-His-His-His-His-His,

Met-His-His-Ala-Gly-Ile-Glu-Gly-Arg and

Met-His-His-Ala-Gly-Asp-Asp-Asp-Asp-Lys.

BRIEF DESCRIPTION OF THE FIGURES

This invention can be more readily understood by reference to thefollowing Description of the Invention and Examples, and to theaccompanying Figures. The following abbreviations and symbols appear inthese Figures:

B, Bg, E, H, N, Na, Nd, P, S, Sa, Sc, X and Xb denote cleavage sites forthe restriction enzymes BamHI, BglII, EcoRI, HindIII, NaeI, NarI, NdeI,PstI, SphI, SAlI, ScaI, XhoI and XbaI, respectively.

represents the promoters of the genes bla, lacI and neo;

represents the ribosomal binding sites of the genes bla, cat, neo andlacI;

represents the terminators t_(o) and T1;

represents the regulatable promoter/operator element P_(N25) x_(/O) orN25OPSN25OP29;

represents the ribosomal binding sites RBSII,SphI and RBSII,3A+5A;→represents the coding regions under control of these ribosomal bindingsites;

represents the regions which code for the affinity peptides inaccordance with the invention as well as the selective cleavage sites;

represents the regions which code for 2.4 or 6 histidine residues;⃡represents the region required for replication (repl.);

represents coding regions for dihydrofolate reductase (dhfr),chloramphenicol acetyltransferase, lac-repressor (lacI), β-lactamase(bla), neomycin phosphotransferase (neo) and the various derivatives ofγ-interferon.

FIG. 1

Schematic representation of plasmid pDS8/RBSII, SphI.

FIGS. 2A-B

Nucleotide sequence of the XhoI/XbaI fragment of plasmidpDS8/RBSII,SphI. This fragment contains the regulatablepromoter/operator element P_(N25) x_(/O), the ribosomal binding siteRBSII,SphI, the dhfr gene, the terminator t_(o), the cat gene and theterminator T1. The cleavage sites for restriction enzymes given in FIG.1 are overlined, while the region being under control of RBSII,SphI,which codes for a variant of dihydrofolate reductase, is underlined. Inaddition, the part of the plasmid pDS8/RBSII,SphI originating fromplasmid pRB322 is shown schematically, with the given numbers referringto the sequence of pBR322 (J.G. Sutcliffe, Cold Spring Harbor Symp.Quant. Biol. 43, pp. 77-90 (1979]).

FIG. 3

Schematic representation of plasmid pDS5/RBSII, 3A+5a.

FIGS. 4A-C

Nucleotide sequence of plasmid pDS5/RBSII,3A+5A. The cleavage sites forrestriction enzymes given in FIG. 3 are overlined, while the regionunder the control of RBSII,3A+5A. which codes for a variant ofchloramphenicol transferase, is underlined.

FIG. 5

Schematic representation of plasmid pDS78/RBSII.

FIGS. 6A-D

Nucleotide sequence of plasmid pDS78/RBSII. The cleavage sites forrestriction enzymes given in FIG. 5 are overlined, while the regionunder the control of RBSII, which codes for a variant of dihydrofolatereductase, is underlined.

FIG. 7

Schematic representation of plasmid pDS56/RBSII.

FIGS. 8A-C

Nucleotide sequence of plasmid pDS56/RBSII. The cleavage sites forrestriction enzymes given in FIG. 7 are overlined, while the regionunder the control of RBSII is underlined.

FIG. 9

Schematic representation of plasmid pDMI,l.

FIGS. 10A-C

Nucleotide sequence of plasmid pDMI,l. The cleavage sites forrestriction enzymes given in FIG. 9 are overlined, while the regionscoding for neomycin phosphotransferase (neo) and lac-repressor (lacI)are underlined.

FIGS. 11A-B

Nucleotide sequences of the oligonucleotides which are employed for theconstruction of the plasmids used in the Examples. In each case two sucholigonucleotides were combined and referred to as the adaptor. Thecleavage sites for the restriction enzymes NaeI, NarI and BglII areoverlined.

FIG. 12

Schematic representation of the construction and isolation offragment 1. This fragment was isolated from the plasmid pRC23/IFI-900and contains the gene for the recombinant human interferon havingCys-Tyr-Cys as the N-terminal amino acids.

FIG. 13

Schematic representation of the construction of the plasmid pGLS byincorporating fragment 1 into plasmid pDS8/RBSII,SphI. In the schematicrepresentation of pGLS, (Sc) denotes the position at which fragment 1has been linked-with plasmid pDS8/RBSII,SphI via the cleavage site forthe restriction enzyme ScaI.

FIG. 14

Schematic representation of the construction and isolation of fragment2. This fragment codes for a human interferon which is shortened at theC-terminus by 8 amino acids and which is referred to as IFN-γ(-8). Inthe given nucleotide sequences the corresponding termination codon isunderlined.

FIG. 15

Schematic representation of the construction of plasmid pIFN-γ(-8) byincorporating fragment 2 into plasmid pDS8/RBSII,SphI via the cleavagesites for restriction enzymes EcoRI and HindIII.

FIG. 16

Schematic representation of the construction and isolation of fragment3. This fragment carries the regulatable promoter/operator elementP_(N25) x_(/O), the ribosomal binding site RBSII,SphI and the adator 3which codes for the amino acid sequenceMet-His-His-Ala-Gly-Ile-Clu-Gly-Arg-Leu-Gly-Ser.

FIG. 17

Schematic representation of the construction of plasmidpDS8/RBSII,SphI-His,His-Xa-BamHI by incorporating fragment 3 intoplasmid pDS8/RBSII,SphI via the cleavage sites for the restrictionenzymes XhoI and BamHI.

FIG. 18

Schematic representation of the construction and isolation of fragment 4which was used in the construction of plasmid pHis,His-Xa-IFN-γ.

FIG. 19

Schematic representation of the isolation of fragment 5 which was usedin the construction of plasmids pHis,His-Xa-IFN-γ, pHis,His-Ek-IFN-γ(-8)and phis, His-Xa-IFN-γ(-8)(Asn).

FIG. 20

Schematic representation of the isolation of fragment 6 which was usedin the construction of plasmid pHis,His-Xa-IFN-γ.

FIG. 21

Schematic representation of the construction of plasmidpHis,His-Xa-IFN-γ by linking fragments 4. 5 and 6. PlasmidpHis,His-Xa-IFN-γ]codes for an IFN-γ fusion protein havingMet-His-His-Ala-Gly-Ile-Glu-Gly-Arg as an additional N-terminal aminoacid sequence (His,His-Xa-IFN-γ).

FIG. 22

Schematic representation of the construction and isolation of fragment 7which was used in the construction of plasmid pHis,His-Ek-IFN-γ(-8).

FIG. 23

Schematic representation of the isolation of fragment 8 which was usedin the construction of plasmids pHis,His-Ek-IFN-γ(-8) andpHis,His-Xa-IFN-γ(-8)(Asn).

FIG. 24

Schematic representation of the construction of plasmidpHis-His-Ek-IFN-γ(-8) by linking fragments 5, 7 and 8. PlasmidpHis,His-Ek-IFN-γ(-8) codes for a IFN-γ fusion protein which isshortened by 8 amino acids and which hasMet-His-His-Ala-Gly-Asp-Asp-Asp-Asp-Lys as an additional N-terminalamino acid sequence (His,His-Ek- -IFN-γ(-8)).

FIG. 25

Schematic representation of the construction and isolation of fragment 9which was used in the construction of plasmidpHis,His-Xa-IFN-γ(-8)(Asn).

FIG. 26

Schematic representation of the construction of plasmidpHis,His-Xa-IFN-γ(-B)(Asn) by linking fragments 5, 8 and 9. This plasmidcodes for a IFN-γ fusion protein which is shortened at the C-terminus by8 amino acids, which is lengthened at the N-terminus by the amino acidsequence Met-His-His-Ala-Gly-Ile-Glu-Gly-Arg and in which additionallyat position 2 the amino acid Asp is replaced by the amino acid Asn(His,His-Xa-IFN-γ(-8)- (Asn)).

FIG. 27

Schematic representation of the construction and isolation of fragment10 which was used in the construction of plasmid p6xHis-DHFR.

FIG. 28

Schematic representation of the construction of plasmid p6xHis-DHFR bylinking fragment 10 with the XhoI/BamHI fragment of plasmid pDS78/RBSIIcontaining the replication region. Plasmid p6xHis-DHFR codes for a DHFRfusion protein having 6 histidines at the N-terminus [(His)₆ -mDHFR].

FIG. 29

Schematic representation of the construction and isolation of fragment11 which was used in the construction of plasmid p4xHis-DHFR.

FIG. 30

Schematic representation of the construction of plasmid p4xHis-DHFR bylinking fragment 11 with the XhoI/BamHI fragment of plasmid pDS78/RBSIIcontaining the replication region. Plasmid p4xHis-DHFR codes for a DHFRfusion protein having 4 histidines at the N-terminus [(His)₄ - MDHFR].

FIG. 31

Schematic representation of the construction and isolation of fragment12 which was used in the construction of plasmid pRBSII-6xHis.

FIG. 32

Schematic representation of the construction of plasmid pRBSII-6xHis bylinking fragment 12 with the XbaI/BamHI fragment of plasmid pDS56/RBSIIcontaining the replication region.

FIG. 33

Schematic representation of the construction and isolation of fragment13 which was used in the construction of plasmid pRBSII-4xHis.

FIG. 34

Schematic representation of the construction of plasmid pRBSII-4xHis bylinking fragment 13 with the XbaI/BamHI fragment of plasmid pDS56/RBSIIcontaining the replication region.

FIG. 35

Schematic representation of the construction and isolation of fragment14 which was used in the construction of plasmid pRBSII-2xHis.

FIG. 36

Schematic representation of the construction of plasmid pRBSII-2xHis bylinking fragment 14 with the XbaI/BamHI fragment of plasmid pDS56/RBSIIcontaining the replication region.

FIG. 37

Schematic representation of the construction of plasmid pDHFR-6xHis bylinking the XbaI/BglII fragment of plasmid pDS78/RBSII containing thereplication region with the BglII/XbaI fragment of plasmid pRBSII/6xHiscontaining the cat gene. Plasmid pDHFR-6xHis codes for a DHFR fusionprotein having 6 histidines at the C-terminus [Met-mDHFR-(His)₆ ].

FIG. 38

Schematic representation of the construction of plasmid pDHFR-2xHis bylinking the XbaI/BglII fragment of plasmid pDS78/RBSII containing thereplication region with the BglII/XbaI fragment of plasmid pRBSII-2xHiscontaining the cat gene. Plasmid pDHFR-2xHis codes for a DHFR fusionprotein having 2 histidines at the C-terminus [Met-MDHFR-(His)₂ ].

FIG. 39

Schematic representation of the construction of plasmidp4xHis-DHFR-4xHis by linking the XbaI/BglII fragment of plasmidp4xHis-DHFR containing the replication region with the BglII/XbaIfragment of plasmid pRBSII-4xHis containing the cat gene. Plasmidp4xHis-DHFR-4xHis codes for a DHFR fusion protein having in each case 4histidines at the N- and at the C-terminus [(His)₄ - mDHFR-(His)₄ ].

FIGS. 40A-B

Nucleotide sequence of the IFN-γ gene coded from plasmid pGLS and theamino acid sequence derived therefrom.

FIGS. 41A-B

Nucleotide sequence of the IFN-γ fusion gene coded from plasmidpHis,His-Xa-IFN-γ and the amino acid sequence derived therefrom.

FIGS. 42A-B

Nucleotide sequence of the IFN-γ fusion gene coded from plasmidpHis,His-Ek-IFN-γ(-8) and the amino acid sequence derived therefrom.

FIGS. 43A-B

Nucleotide sequence of the IFN-γ fusion gene coded from plasmidpHis,His-Xa-IFN-γ(-8)(Asn) and the amino acid sequence derivedtherefrom.

DESCRIPTION OF THE INVENTION

The affinity peptides can be linked directly or indirectly to thebiologically active Polypeptide or protein. When a single affinitypeptide is used, it can be linked either to the amino-terminal aminoacid or to the carboxy-terminal amino acid of the biologically activePolypeptide or protein. When two affinity peptides are used, one of themis linked to the amino-terminal amino acid of the biologically activepolypeptide or protein and the other is linked to the carboxy-terminalamino acid.

In the case of indirect linking, the affinity peptides contain asuitable selective cleavage site via which they are linked to thedesired biologically active polypeptide or protein. Preferred selectivecleavage sites have the amino acid sequence -(Asp)_(n) - Lys-, wherein nsignifies 2, 3 or 4. or -Ile-Glu-Gly-Arg-, which are specificallyrecognized by the proteases enterokinase and coagulation factor X_(a),respectively. Such affinity peptides can then be cleaved enzymatically,using methods known in the art.

In the case of direct linking, the affinity peptides remain linked withthe desired biologically active polypeptide or protein and cannot bechemically or enzymatically cleaved. This type of linking isadvantageous when the activity of the desired polypeptide or protein isnot impaired by the presence of the affinity peptide. Such fusionproteins can be used for a number of immunological procedures. They canbe used, for example, as reagents for detecting infectious diseases.Because they can be admixed with a physiologically compatible carriermaterial, they can also be used as vaccines for the prevention ofdiseases.

The term "biologically-active polypeptide or protein" as used hereinmeans polypeptides or proteins which themselves are biologically active,or polypeptides or proteins which can be used for the preparation ofbiologically active polypeptides or proteins.

Biologically active polypeptides or proteins which can be used in thisinvention include, for example, malaria surface antigens, especially the5.1 surface antigen, the CS protein and the p190 protein of Plasmodiumfalciparum, lymphokines, interferons, insulin and insulin precursors,HIV-1 and HIV-2 envelope and structural proteins, growth hormones andgrowth hormone releasing factors. Especially preferred biologicallyactive polypeptides or proteins are those having the amino acid sequenceof human immune interferon and partial sequences of human immuneinterferon, especially those having the amino acid sequences of theformulae:

Gln-Asp-Pro-Tyr-Val-Lys-Glu-Ala-Glu-Asn-Leu-Lys-Lys-Tyr-Phe-Asn-Ala-Gly-His-Ser-Asp-Val-Ala-Asp-Asn-Gly-Thr-Leu-Phe-Leu-Gly-Ile-Leu-Lys-Asn-Trp-Lys-Glu-Glu-Ser-Asp-Arg-Lys-Ile-Met-Gln-Ser-Gln-Ile-Val-Ser-Phe-Tyr-Phe-Lys-Leu-Phe-Lys-Asn-Phe-Lys-Asp-Asp-Gln-Ser-Ile-Gln-Lys-Ser-Val-Glu-Thr-Ile-LYS-Glu-Asp-Met-Asn-Val-Lys-Phe-Phe-Asn-Ser-Asn-Lys-Lys-Lys-Arg-Asp-Asp-Phe-Glu-Lys-Leu-Thr-Asn-Tyr-Ser-Val-Thr-Asp-Leu-Asn-Val-Gln-Arg-Lys-Ala-Ile-His-Glu-Leu-Ile-Gln-Val-Met-Ala-Glu-Leu-Ser-Pro-Ala-Ala-Lys-Thr-Gly-Lys-Arg-Lys-Arg-Ser-Gln-Met-Leu-Phe-Arg-Gly-Arg-Arg-Ala-Ser-Gln.

Gln-Asp-Pro-Tyr-Val-Lys-Glu-Ala-Glu-Asn-Leu-Lys-Lys-Tyr-Phe-Asn-Ala-Gly-His-Ser-Asp-Val-Ala-Asp-Asn-Gly-Thr-Leu-Phe-Leu-Gly-Ile-Leu-Lys-Asn-Trp-Lys-Glu-Glu-Ser-Asp-Arg-Lys-Ile-Met-Gln-Ser-Gln-Ile-Val-Ser-Phe-Tyr-Phe-Lys-Leu-Phe-Lys-Asn-Phe-Lys-Asp-Asp-Gln-Ser-Ile-Gln-Lys-Ser-Val-Glu-Thr-Ile-Lys-Glu-Asp-Met-Asn-Val-Lys-Phe-Phe-Asn-Ser-Asn-Lys-Lys-Lys-Arg-Asp-Asp-Phe-Glu-Lys-Leu-Thr-Asn-Tyr-Ser-Val-Thr-Asp-Leu-Asn-Val-Gln-Arg-Lys-Ala-Ile-His-Glu-Leu-Ile-Gln-Val-Met-Ala-Glu-Leu-Ser-Pro-Ala-Ala-Lys-Thr-Gly-Lys-Arg-Lys-Arg-Ser-Gln-Met-Leuand

Gln-Asn-Pro-Tyr-Val-Lys-Glu-Ala-Glu-Asn-Leu-Lys-Lys-Tyr-Phe-Asn-Ala-Gly-His-Ser-Asp-Val-Ala-Asp-Asn-Gly-Thr-Leu-Phe-Leu-Gly-Ile-Leu-Lys-Asn-Trp-Lys-Glu-Glu-Ser-Asp-Arg-Lys-Ile-Met-Gln-Ser-Gln-Ile-Val-Ser-Phe-Tyr-Phe-Lys-Leu-Phe-Lys-Asn-Phe-Lys-Asp-Asp-Gln-Ser-Ile-Gln-Lys-Ser-Val-Glu-Thr-Ile-Lys-Glu-Asp-Met-Asn-Val-Lys-Phe-Phe-Asn-Ser-Asn-Lys-Lys-Lys-Arg-Asp-Asp-Phe-Glu-Lys-Leu-Thr-Asn-Tyr-Ser-Val-Thr-Asp-Leu-Asn-Val-Gln-Arg-Lys-Ala-Ile-His-Glu-Leu-Ile-Gln-Val-Met-Ala-Glu-Leu-Ser-Pro-Ala-Ala-Lys-Thr-Gly-Lys-Arg-Lys-Arg-Ser-Gln-Met-Leu,

and those having the amino acid sequence of mouse dihydrofolatereductase.

The preparation of the fusion proteins of this invention can be carriedout using standard recombinant DNA methods. Preferably, a nucleotidesequence coding for the desired affinity peptide is first synthesizedand then linked to a nucleotide sequence coding for the desiredbiologically active polypeptide or protein.

The thus-obtained hybrid gene can be incorporated-into expressionvectors such as plasmid pDS8/RBSII, SphI; pDS5/RBSII,3A+5A; pDS78/RBSII;pDS56/RBSII or other commercial or generally accessible plasmids, usingstandard methods. Most of the requisite methodology can be found inManiatis et al., "Molecular Cloning", Cold Spring Harbor Laboratory,1982, which is hereby incorporated by reference to illustrate the stateof the art.

Methods for the expression of the fusion proteins of this invention arealso described by Maniatis et al., supra. They embrace the followingprocedures:

(a) Transformation of a suitable host organism, preferably E. coli, withan expression vector in which the hybrid gene is operatively linked toan expression control sequence;

(b) Cultivation of the transformed host organism under suitable growthconditions; and

(c) Extraction and isolation of the desired fusion protein from the hostorganism.

Host organisms that can be used include but are not limited togram-negative and gram-positive bacteria such as E. coli and B. subtilisstrains. E. coli strain M15 is especially preferred. Other E. colistrains that can be used include, e.g., E. coli 294 (ATCC No. 3144), E.coli RR1 (ATCC No. 31343) and E. coli W3110 (ATCC No. 27325).

Ideal metal chelate resins for the purification of the fusion proteinsof the invention are nitrilotriacetic acid (NTA) resins of the generalformula:

Carrier matrix-spacer--NH--(CH₂)_(x) --CH(COOH)----N(CH₂ COO⁻)₂ Ni²⁺,wherein x signifies 2. 3 or 4.

Carrier matrices that can be used include, e.g.. materials used inaffinity and gel chromatography such as cross-linked dextrans, agarose(especially in the form known under the trademark Sepharose®) orpolyacrylamides.

Suitable spacer groups include these known in the field of affinitychromatography, with the groups --O--CH₂ --CH(OH)--CH₂ -- and --O--CO--being preferred.

An especially preferred NTA resin for the purification of the hybridproteins of this invention has the formula: ##STR1##

The NTA resin can be used batch-wise or in continuously operatingcolumns to purify the fusion proteins. Prior to loading with the fusionprotein, the NTA resin is equilibrated with an aqueous buffer whichitself does not form chelates with nickel, preferably a Tris.HCl buffer.pH 7.5. The equilibration buffer (and the elution buffer) can contain adenaturing agent or a detergent such as guanidine.HCl, urea or Triton.The addition of such a denaturing agent or detergent permitsproblem-free operations even with fusion proteins which are poorlysoluble in aqueous solution.

The elution of the fusion proteins from the column can be carried out ata constant pH or with linear or discontinuously falling pH gradients.The optimal elution conditions depend on the amount and type ofimpurities which are present, the amount of material to be purified, thecolumn dimensions etc. and are easily determined by routineexperimentation on a case-by-case basis.

EXAMPLES

The following Examples illustrate the preparation of fusion proteins ofthe invention, their purification by means of metal chelatechromatography and the preparation of biologically active polypeptidesor proteins by enzymatic cleavage of the purified fusion proteins inaccordance with the invention.

Example 1 Description of Plasmids Used In the Construction of PlasmidspGLS, pHis,His-Xa-IFN-γ, P-His,His-Ek-IFN-γ(-8),pHis,His-Xa-IFN-γ(-8)(Asn), p6xHis-DHFR, p4xHis-DHFR-4xHis, pDHFR-2xHisand pDHFR-6xHis A. Principles

Plasmids pDS8/RBSII,SphI (FIGS. 1 and 2). pDS5/RBSII,3A+5A (FIGS. 3 and4A-C), pDS78/RBSII (FIGS. 5 and 6A-D) and pDS56/RBSII (FIGS. 7 and 8A-C)were used for the construction of the specified plasmids. E. coli cellstransformed with these plasmids were deposited at the Deutsche Sammlungvon Microorganism in Grisebachstrasse 8, D-3400 West Germany Gottingen(on Nov. 21, 1987 in Braunschweig) on Oct. 3. 1985 [E. coli M15(pDS5/RBSII,3A+SA; pDMI,1), DSM No.: 3517], on Aug. 6, 1986 (E. coli M15(pDS8/ RBSII,SphI; pDMI,1), DSM No.: 3809], on Sep. 3, 1987 (E. coli M15(pDS78/RBSII; pDMI,1). DSM No.: 4232). and on Dec. 23, 1987 (E. coli M15(pDS56/RBSII; pDMI,1), DSM No.: 4330]. in accordance with the BudapestTreaty.

The above-mentioned vectors contain the regulatable promoter/operatorelement P_(N25) x_(/O) (Stuber et al., EMBO J. 3. 3143-3148 (1984)) orN25OPSN25OP29 and the ribosomal binding sites RBSII,SphI, RBSII,3A+5A orRBSII. These ribosomal binding sites were derived from the ribosomalbinding site of the promoter PG25 of the E. coli phage T5 (R. Gentz,Dissertation, Universitat Heidelberg, BRD [1984]) and were obtained viaDNA synthesis.

The high efficiency of expression of the above-mentioned plasmids can bemaintained in E. coli cells only if the promoter/operator element isrepressed by the binding of a lac repressor to the operator. The lacrepressor is coded in the lacI gene. P_(N25) x_(/O) and N25OPSN25OP29can be repressed efficiently only when a sufficient number of repressormolecules is present in the cells. Therefore, the lacI^(q) allel, whichcontains a promoter mutant which leads to an increased expression of therepressor gene, was used. This lacI^(q) allel is present in the plasmidpDMI,l (FIGS. 9 and 10A-C).

This plasmid carries, in addition to the lac-I gene, the neo gene, whichconfers kanamycin resistance to the bacteria and which is used as theselection marker. pDMI,l is compatible with the aforementioned plasmids.E. coli cells which are transformed with such expression vectors mustcontain pDMI,l to guarantee that the expression vector is held stable inthe cells. An induction of this system is achieved by adding IPTG to themedium at the desired cell density.

B. Plasmid pDS8/RBSII,SDhI

The part of pDS8/RBSII,SphI (FIGS. 1 and 2A-B) which lies between therestriction cleavage sites for XbaI and XhoI and which contains thereplication region and the gene for βlactamase (which confers ampicillinresistance to the cells) was derived from the plasmid pBR322 (Bolivar etal., Gene 2, 95-113 [1977]); Sutcliffe, Cold Spring Harbor Symp. Quant.Biol. 43, 77-90 [1979]). The remaining part of the plasmid carries theregulatable promoter/operator element P_(N25) x_(/O) (Stuber et al.,supra) followed by the ribosomal binding site RBSII, SphI, which is partof a EcoRI/BamHI fragment; the dihydrofolate reductase (DHFR) gene ofmouse cell line AT-3000 (Chang et al., Nature 275, 617-624 (1978);Masters et al., Gene 21, 59-63 [1983]); the terminator t_(o) of E. coliphage lambda (Schwarz et al., Nature 272, 410-414 [1978]); thepromoter-free gene of chloramphenicol acetyl-transferase (Marcoli etal., FEBS Letters, 110, 11-14 [1980]) and the terminator T1 of the E.coli rrnB operon (Brosius et al., J. Mol. Biol., 148, 107-127 (19811).

C. Plasmid pDS5/RBSII,3A+5A

The part of pDS5/RBSII,3A+5A (FIGS. 3 and 5A-C) which lies between thecleavage sites for the restriction enzymes XbaI and XhoI and whichcontains the replication region and the gene for β-lactamase (whichconfers ampicillin resistance to the cells) was derived originally fromthe plasmid pBR322 (Bolivar et al., supra; Sutcliffe, supra). However,the gene for β-lactamase is modified by elimination of the cleavagesites for the restriction enzymes HincII and PstI. These alterations inthe DNA sequence have no effect on the amino acid sequence of theβ-lactamase. The remaining part of the plasmid carries the regulatablepromoter/operator element P_(N25) x_(/O) (Stuber et al., supra) followedby the ribosomal binding site RBSII,3A+5A, which is part of aEcoRI/BamHI fragment; cleavage sites for the restriction enzymesSalI,PstI and HindIII; the promoter-free gene for chloramphenicolacetyltransferase (Marcoli et al., supra) and the terminator T1 of theE. coli rrnB operon (Brosius et al., supra).

D. Plasmid pDS78/RBSII

The part of pDS78/RBSII (FIGS. 5 and 6A-D) which lies between therestriction cleavage sites for XbaI and XhoI and which contains thereplication region and the gene for β-lactamase (which confersampicillin resistance to the cells) was derived originally from theplasmid pBR322 (Bolivar et al., supra; Sutcliffe, supra). However, thegene for β-lactamase is modified in the manner described for the plasmidpDS5/RBSII,3A+5A. The remaining part of the plasmid carries theregulatable promoter/operator element N25OPSN25OP29 followed by theribosomal binding site RBSII, which is part of a EcoRI/BamHI fragment;the gene for dihydrofolate reductase of the mouse cell line AT-3000(Chang et al., supra; Masters et al., supra), which has been altered byintroducing a cleavage site for the restriction enzyme BglII immediatelyprior to the end of the structural gene; the terminator t_(o) (Schwarzet al., supra); the promoter-free gene for chloramphenicolacetyltransferase (Marcoli et al., supra) and the terminator T1 (Brosiuset al., supra).

E. Plasmid pDS56/RBSII

Plasmid pDS56/RBSII (FIGS. 7 and 8A-C) is very similar to plasmidpDS5/RBSII,3A+5A but contains the regulatable promoter/operator elementN25OPSN25OP29 and the ribosomal binding site RBSII as expressionsignals. In addition, pDS56/RBSII contains the terminator t_(o) of theE. coli phage lambda (Schwarz et al., supra).

F. Plasmid pDMI,l

Plasmid pDMI,l (FIGS. 9 and 10A-C) carries the gene for neomycinphosphotransferase from the transposon Tn5 (Beck et al., Gene 19,327-336 [1382]), which confers kanamycin resistance to the E. colicells, and the lacI gene (Farabough, Nature 274, 765-769 [1978]) withthe promoter mutation I¹ (Calos, Nature 274, 762-765 [1978]), whichcodes for the lac repressor. Moreover, plasmid pDMI,l contains a regionof the plasmid pACYC184 (Chang and Cohen, J. Bacteriol. 134, 1141-1156[1978]), which contains all information required for the replication andstable transmission to the daughter cells.

Example 2 Description of DNA Adapters Used In the Construction of theVarious Plasmids A. Principles

To adapt the ribosomal binding site RBSII, SphI on the gene for immuneinterferon (IFN-γ), to shorten this gene, to link IFN-γ and IFN-γfragments such as, e.g., IFN-γ(-8) with an affinity peptide and toexpress DHFR fusion proteins having at least two neighbouring histidineresidues, oligonucleotides were chemically synthesized and, afterworkup, phosphorylated. The nucleotide sequences of the adaptors usedare shown as double-stranded DNA sequences in FIGS. 11A-B.

B. Synthesis and Workup of the Oligonucleotides

The oligonucleotides were prepared simultaneously on a multisynthesisapparatus (described in European Patent Application No. 181, publishedMay 21, 1985), with glass of defined pore size (CPG) used as the carriermaterial (Kiefer et al., Immuno. Meth. 3, 69-83 [1985]; Sproat et al.,Tetrahedr. Lett. 24 5771-5774 [1983]; Adams et al., J. Amer. Chem. Soc..105, 661-663 [1985]. The lyophilized oligonucleotides were taken up inwater and dissolved at 4° C. for 1 hour. The DNA concentration was 100nmoles/ml.

C. Phosphorylation of the Oligonucleotides

In each case, 150 pmol of the oligonucleotides were incubated inseparate batches at 37° C. for 20 minutes in 20 μl of 50 mM Tris HCl, pH8.5, and 10 mM MgCl₂ with 2 pmol of γ[³² P]-ATP (Amersham, Braunschweig;5000 Ci/mmol) and 1 unit (U) of T₄ polynucleotide kinase (Gibco-BRL,Basle). Subsequently, 5 nmol of ATP were added, and after a further 20minutes at 37° C., the reaction was terminated by heating to 65° C. Thephosphorylated oligonucleotides produced were used without any furtherworkup.

Example 3 Construction of Plasmid pGLS A. Principles

For the construction of plasmid pGLS, the IFN-γ gene was first bonded(FIG. 12) to adaptor 1 (FIGS. 11A-B) and isolated. Subsequently, theresulting fragment 1 was integrated into plasmid pDS8/RBSII,SphI (FIG.13).

B. Preparation of Fragment 1

4 μg of plasmid pRC23/IFI-900 (European Patent Application publicationNo. 99084, published Jan. 25, 1984) having a DNA concentration of 400μg/ml were digested with 10 units of the restriction endonuclease NdeIin core buffer (50 mM Tris HCl, pH 8, 10 MM MgCl₂, 50 mM NaCl) for 1hour at 37° C. (volume 20 μl). The sample was subsequently extractedonce with phenol, the remainder of the phenol was removed with ether andthe DNA was finally precipitated with 66% alcohol and 0.3M potassiumacetate. The sediment was dried for 2 minutes in a Speed-vacconcentrator and dissolved in T4 ligase buffer (50 mM Tris HCl, pH 7.8,10 mM MgCl₂, 10 mM DTT, 500 μm ATP).

25 pmol of the phosphorylated adaptor 1 (FIGS. 11A-B) were dissolved inlx ligase buffer and added to this reaction mixture so that a totalvolume of 25 μl was achieved. The ligation was carried out for 3 hoursat 22° C., with 1 μl of DNA ligase (1 White unit, Boehringer Mannheim)being used. The ligation was terminated by heating the sample to 65° C.for 7 minutes.

The DNA was precipitated with alcohol, dried as described above and thendissolved in 50 μl of NcoI digestion buffer (50 mM Tris HCl, pH 8, 10 mMMgCl₂, 50 mM NaCl. 50 mM KCl). 10 units of NcoI were added thereto andthe sample was incubated for 1 hour at 37° C. The enzyme wassubsequently inactivated by heating the sample to 650° C. for 7 minutes.

After a phenol extraction the DNA was precipitated as described aboveand the sediment was dried. The DNA was dissolved in Klenow buffer (50mM Tris HCl, pH 7.2, 10 mM MgSO₄, 100 μM DTT), dATP, dGTP, dCTP and dTTP(final concentration in each case 100 μM) and 1 unit of Klenow enzyme(Boehringer, Mannheim) were added thereto and the sample was held for 1hour at 22° C. The reaction was terminated by the addition of 2 μl of0.25 M EDTA, the sample was extracted with phenol, the DNA wasprecipitated with alcohol as described and dissolved in SphI digestionbuffer (50 mM Tris HCl, pH 7.5, 6 mM MgCl₂, 50 mM NaCl, 6 mM2-mercaptoethanol).

After the addition of 10 units of SphI the sample was incubated for 1hour at 37° C. the digestion was terminated by heating as described, aphenol extraction was carried out and the DNA was precipitated withalcohol and dried.

The DNA sediment was dissolved in 10 μl of sample buffer and the DNAfragments were separated in a 6% polyacrylamide gel (elution buffer; 40mm Tris HCl, 20 mM Na acetate, 2 mM EDTA, pH 7.8). DNA of phage φX(Gibco-BRL, Basle) digested with HaeIII was used as the molecular weightstandard. The DNA was stained with ethidium bromide (0.5 μg/ml), madevisible with UV light (300 nm wavelength). and the IFN-γ coding band wascut out from the gel with a scalpel.

The piece of gel was transferred into a pocket of size 4×4 mm in anagarose gel (0.7% agarose gel, running buffer: 90 mM Tris borate, 90 mMboric acid, 3 mM EDTA, pH 8.3). The pocket was closed with 0.7% liquidagarose in lxTBE in order to achieve a homogeneous electrical field. Aportion of a NA45 membrane (Schleicher and Schull, Dassel, BRD) wasplaced in front of the sample and the DNA was electrophoretized onto themembrane (5 minutes, 15 V/cm).

After washing with distilled water, the membrane was transferred into anEppendorf test tube which contained 250 μl of 1.5M lithium acetate, 50mM Tris HCl, pH 8, and 10 mM EDTA. The DNA was eluted for 20 minutes at65° C. The membrane strip was removed from the test tube and the samplewas extracted once with 200 μl of phenol (pH 8). The DNA wasprecipitated after the addition of 20 μl of 5M lithium acetate and 440μl of isopropanol, and the sediment was washed with 80% ethanol anddried. Subsequently, the sediment was dissolved in 10 μl of TE buffer(10 mM Tris HCl, pH 7.6, 1 mM EDTA). The DNA fragment produced wasdesignated fragment 1 (FIG. 12).

C. Preparation of Plasmid pDS8/RBSII,SphI

2 pmol of plasmid pDS8/RBSII,SphI were cleaved with the restrictionenzyme SphI. Thereafter, the resulting linear plasmid DNA was incubatedwith a limited amount of the restriction enzyme ScaI, whereby the DNAwas cleaved only at approximately 50% of the ScaI cleavage sitespresent. The sample was extracted with phenol and then with ether, andthe DNA was precipitated as described above. The sediment was dried anddissolved in 20 μl of buffer (5 mM Tris HCl, pH 8), and 1 U of CIP (calfintestinal phosphatase, Boehringer Mannheim) was added.

The sample was incubated for 1 hour at 37° C., the enzyme was removed bya phenol extraction and the DNA was precipitated. After dissolving theDNA, the ScaI/SphI fragment, which contained part of the cat gene, theterminator T1, the replication region, the bla gene, the promoter N25x/oand the ribosomal binding site RBSII,SphI, was isolated from a 1%agarose gel and transferred electrophoretically to a NA45 membrane asdescribed above. Elution of the DNA, alcohol precipitation anddissolution in 10 μl of TE buffer were also carried out as describedabove. Approximately 1 pmol of the desired vector fragment was obtained.

D. Assembly of Plasmid pGLS

0.05 pmol of the pDS8/RBSII, SphI vector fragment were ligated asdescribed above with 0.05 pmol of insert DNA (fragment 1) in ligasebuffer. A control ligation without insert DNA was carried out inparallel. E. coli M15 cells containing plasmid pDMI,l were prepared forthe transformation according to the method of Morrison (Methods Enzymol.68, 326-331 [1979]). After heating for 7 minutes at 65° C., the ligationmixtures were added to 200 μl of these competent cells. The samples weremaintained for 30 minutes on ice, incubated for 2 minutes at 420° C.and, after the addition of 0.5 ml of LB medium. incubated for 90 minutesat 370° C.

The cells were then plated-out on LB agar plates which contained 100μg/ml ampicillin and 25 μg/ml kanamycin and incubated overnight at 37°C. The transformation of the control ligation gave no transformants. Theligation with fragment 1 on the other hand gave about 200 colonies.

Individual colonies were picked with a sterile toothpick, transferredinto a test tube which contained 10 ml of LB medium with 100 μg/mlampicillin and 25 μg/ml kanamycin and maintained for 12 hours in ashaking incubator. Thereafter, the cells were sedimented and the plasmidDNA was isolated according to the method of Birnboim and Doly (NucleicAcids Res. 7. 1515-1523 (1979]).

In each case, 1 μg of the isolated plasmids was digested with SphI andXbaI to determine whether a fragment which contained the IFN-γ gene andthe terminator T1 was present in these plasmids. All analyzed DNAsamples contained the mentioned DNA fragment of about 1 kb. Theseplasmids were designated pGLS (FIG. 13).

E. Sequence Analysis of the IFN-γ Gene Integrated Into pGLS

To determine whether the correct IFN-γ sequence is present in plasmidpGLS, the double-stranded circular plasmid DNA was sequenced, with astarter sequence (primer) labelled with [γ-³² P]-ATP being used. Thisstarter sequence contains the nucleotides of position 199-218 of theplasmid pDS8/RBSII,SphI and ends 6 nucleotides before the ATG of theSphI cleavage site.

0.3 pmol of the isolated plasmid DNA was precipitated with alcohol, thesediment was washed once with 80% ethanol, dried and finally dissolvedin 8 μl of 1/4 TE buffer. After the addition of 2 pmol of the startersequence, the sample was incubated for 5 minutes at 42° C. The DNA wasthen sequenced according to the method of Sanger et al. (Proc. Natl.Acad. Sci. USA 74, 5463-6567 1977]). As a radioactively labelled"primer" was used, all reactions were carried out with unlabelleddeoxynucleotide triphosphates. The DNA sequence analysis indicated thatthe correct IFN-γsequence had been integrated into plasmid pGLS (foramino acid sequence, see FIG. 40).

Example 4 Construction of Plasmid pIFN-γ(-8) A. Principles

For the construction of plasmid pIFN-γ(-8), adaptor 2 (FIGS. 11A-B) wasfirst linked to the individual HinfI cleavage site in the IFN-γ gene.Because of a translation stop codon in this adaptor, the C-terminalregion of the IFN-γ protein is shortened by 8 amino acids (FIG. 14). Theresulting fragment 2 was subsequently integrated into plasmidpDS8/RBSII,SphI (FIG. 15).

B. Preparation of Fragment 2

3 pmol of plasmid pGLS were digested with 15 U of HinfI (50 μl volume, 1hour, 37° C.). The restriction enzyme was subsequently inactivated (7min. at 65° C.), the sample was extracted with phenol, extracted withether. precipitated with potassium acetate and alcohol and dried.

The sediment was dissolved in 50 μl of ligase buffer. 100 pmol ofphosphorylated oligonucleotide (adaptor 2) were mixed with 10 μl ofHinfI-cleaved plasmid pGLS and, after the addition of 25 μl of ligasebuffer and 1 U of T4-DNA ligase, incubated for 12 hours at 22° C. Asdescribed above, the reaction was terminated by heating the sample andthe DNA was precipitated.

A digestion with the restriction enzymes EcoRI (15U) and HindIII (20U)for 20 hours at 37° C. was then carried out. After heat inactivation,phenol extraction, extraction with ether and alcohol precipitation, thesample was dissolved in 10 μl of sample buffer and the DNA fragmentswere separated in a 6% polyacrylamide gel. After staining with ethidiumbromide the DNA bands were made visible under UV light (300 nm). Theband which contained the IFN-γ gene was cut out from the gel with asterile scalpel and electrophoresed as described above onto a NA45membrane. The DNA was eluted and designated fragment 2 (FIG. 14).

C. Preparation of Plasmid DDS8/RBSII,SphI

2 pmol of plasmid pDS8/RBSII,SphI were digested with 10 U of EcoRI and10 U of HindIII for 1 hour at 37° C., in a volume of 50 μl. After heatinactivation of the enzyme and acohol precipitation the DNA sediment wasdissolved in 10 μl of sample buffer. After electrophoreses in a 1%agarose gel the EcoRI/HindIII fragment which contained the terminatort_(o), the cat gene, the terminator T1, the replication region, the blagene and the promoter P_(N25) x_(/O) was cut out from the gel and elutedas described above.

D. Assembly of Plasmid pIFN-γ(-8)

10 μl of the isolated EcoRI/Hind III vector fragment and half of theisolated fragment 2 were incubated (20° C. 3 hours) with 1 U of T4ligase. A control ligation without the addition of fragment 2 wascarried out in parallel. The ligations were terminated by heating thesamples as previously described.

The transformations were carried out according to the method of Morrison(supra), with E. coli strain M15, which contained the plasmid pDMI.l,being used. The cells were plated-out on LB agar plates which contained100 μg/ml ampicillin and 25 μg/ml kanamycin. The plates were held for 15hours at 370° C. in an incubator.

No transformants were found on the control plates, but the ligation inwhich the vector DNA and fragment 2 were used gave about 500 colonies.Individual colonies were picked with a sterile toothpick, transferredinto 100 ml of LB medium and left to grow as described. The plasmid DNAwas isolated according to the method of Birnboim and Doly (supra). Ineach case 4 μl of the plasmid DNA, is isolated and dissolved in TEbuffer, were cleaved as described above with 2 U of EcoRI and HindIII. Afragment with the desired length of about 450 bp could be cut out fromall tested plasmids. These plasmids were designated pIFN-γ(-8) (FIG.15).

E. Sequence Analysis of Plasmid pIFNγ(-8)

Sequence analysis was carried out as described in Example 3. However, anoligonucleotide which contained the nucleotides of position 928-896 ofthe plasmid pDS8/RBSII,SphI and which therefore permitted the sequencingof DNA fragments which were integrated in front of the terminator t_(o)was used as the starter sequence. The sequence analysis confirmed thedesired sequence of IFN-γ gene which codes for a IFN-γ protein shortenedby 8 amino acids (at the carboxyl end).

Example 5 Construction of Plasmid pDS8/RBSII,SphI-His,His-Xa-BamHI A.Principles

For the construction of plasmid pDS8/RBSII,SphI-His,His-xa-BamHI,adaptor 3 (FIGS. 11A-B), which codes for an affinity peptide whichcontains two neighbouring histidines and a cleavage site of factor Xawas linked with the ribosomal binding site RESII,SphI. Subsequently,fragment 3 (FIG. 16) containing the promoter P_(N25) x_(/O) and the saidaffinity peptide was isolated and integrated into plasmidpDS8/RBSII,SphI (FIG. 17).

B. Preparation of Fragment 3

2 pmol of plasmid pDS8/RBSII,SphI were cleaved with the restrictionenzyme SphI. The reaction was terminated by incubation at 65° C. for 7minutes, the sample was extracted with phenol, extracted with ether andthe DNA was precipitated with alcohol and potassium acetate. Thesediment was taken up in 10 μl of ligase buffer, 25 pmol ofphosphorylated adaptor 3 (FIGS. 11A-B) dissolved in ligase buffer wereadded, and after the addition of 1 U of T4 DNA ligase, incubated for 3hours at 22° C. The reaction was terminated by heating (7 minutes. 65°C.). and after a phenol extraction and subsequent treatment with ether,the DNA was precipitated with alcohol and potassium acetate. Thesediment was dissolved in 30 μl of buffer, in each case 10 U of therestriction enzymes BamHI and XhoI were added and the mixture wasincubated at 37° C. for 2 hours. Subsequently, 3.5 μl of 10 -foldconcentrated sample buffer for polyacrylamide gels-were added to thesample and the mixture was incubated for 7 minutes at 65° C. The DNA wasseparated in a 6% polyacrylamide gel, and the fragment liberated by XhoIand BamHI was cut out with a scalpel. The DNA was eluted as describedand designated fragment 3 (FIG. 16).

C. Preparation of Plasmid pDS8/RBSII,SphI

2 pmol of plasmid pDS8/RBSII,SphI were cleaved with 10 U each of therestriction enzymes BamHI and XhoI. After heat inactivation of theenzyme, the sample was extracted with phenol, extracted with ether andthe DNA was precipitated with alcohol and potassium acetate. Thesediment was re-suspended in 50 μl of 50 mM Tris HCl, pH 8. 1 U of CIP(see above) was added, and the sample was o incubated at 37° C. for 30minutes. After heat inactivation of the enzyme, the DNA was separated ina 6% polyacrylamide gel after the addition of sample buffer, and theplasmid body was eluted from the gel as described.

D. Assembly of Plasmid pDS8/RBSII,SphI-His,His-Xa-BaMHI

The above-described fragment 3 was ligated with the vector body (22° C.,2 U of T4 DNA ligase, 25 μl of ligase buffer). A control ligationwithout the addition of fragment 3 was carried out in parallel. Theligation batches were transformed as described above into E. coli strainM15 which contained the plasmid pDMI.l and plated out on LB plates with100 μg/ml ampicillin and 25 μg/ml kanamycin. The transformation of thecontrol ligation gave no transformants but the transformation of theligation batch with fragment 3 gave about 100 colonies.

Individual colonies were grown up in 100 ml of LB medium as describedabove and the plasmid DNA was isolated according to the method ofBirnboim and Doly (supra). All plasmids contained the cleavage sites forNaeI and NarI (see FIG. 17) newly introduced by the adaptor. Sequenceanalysis of the plasmid DNA carried out as described above (Example 3,E) confirmed that adaptor 3 had been correctly integrated into thevector. These plasmids were designated pDS8/RBSII,SphI-His,His-Xa-BamHI(FIG. 17).

Example 6 Construction of Plasmid pHis,His-Xa-IFN-γ A. Principles

For the construction of plasmid pHis,His-Xa-IFN-γ, the following DNAfragments were isolated and linked with one another (FIG. 21): 1) theIFN-γ gene of plasmid pGLS (fragment 4, FIG. 18) linked with adaptor 4(FIGS. 11A-B): 2) the signal unit of the plasmidpDS8/RBSII,SphI-His,His-Xa-BamHI, which contains the promoter P_(N25)x_(/O), the ribosomal binding site RBSII, SphI and the region coding forthe neighbouring histidines and for the recognition site of factor Xa(fragment 6, FIG. 20); and 3) the replication region with theβ-lactamase gene from plasmid pDSS/RBSII,3A+5A (fragment 5, FIG. 19).

B. Preparation of Fragment 4

2 pmol of plasmid pGLS were cleaved with the restriction enzyme NdeI.After heat inactivation of the enzyme, the sample was extracted withphenol, extracted with ether and the DNA was precipitated as described.The sediment was dissolved in 10 μl of ligase buffer. 50 pmol ofphosphorylated adaptor 4 (FIGS. 11A-B) dissolved in ligase buffer wereadded to the NdeI-cleaved plasmid pGLS, and the sample was incubated(22° C. 3 hours) with 2 U of ligase. After heat inactivation of theligase, the sample was extracted with phenol, extracted with ether andthe DNA was precipitated as described. The sediment was dissolved andthe DNA was cleaved with the restriction enzymes NarI and XbaI. Afterthe addition of sample buffer, heating the mixture at 65° C. for 7minutes and separation of the DNA in a 6% polyacrylamide gel theNarI/XbaI fragment, which contained the IFN-γ gene, was isolated asdescribed above. This fragment was designated fragment 4 (FIG. 18).

Preparation of Fragment 5

2 pmol of plasmid pDS5/RBSII,3A+5A were cleaved with the restrictionenzymes XhoI and XbaI. The mixture was worked-up as described above, andthe DNA was separated in a 6% polyacrylamide gel. The fragment whichcontained the bla gene and the replication region was isolated from thegel as described. This fragment was designated fragment 5 (FIG. 19).

D. Preparation of Fragment 6

2 pmol of plasmid pDS8/RBSII,SphI-His,His-Xa-BamHI were cleaved with therestriction enzymes XhoI and NarI. After working-up the sample and gelelectrophoresis, fragment 6, which contains the promoter P_(N25) x_(/O),the ribosomal binding site RESII,SphI and the region which codes for theneighbouring histidines and the recognition site for factor Xa (FIG. 20)was isolated.

E. Assembly of Plasmid pHis,His-Xa-IFN-γ

0.5 pmol each of fragments 4,(FIG. 18). 5 (FIG. 19) and 6 (FIG. 20) wereincubated (220° C. 5 hours) in ligase buffer with 2 U of T4 DNA ligase.After heat inactivation of the enzyme, the batch was transformed asdescribed above into E. coli strain M15 which contained the plasmidpDMI.l, and the transformation mixture was plated-out on LB agar plateswhich contained 100 μg/ml ampicillin and 25 μg/ml kanamycin.

After incubation at 370° C. overnight, about 100 transformants wereobtained. Individual colonies were grown up in 100 ml of LB medium aspreviously described, and the plasmids were isolated according to themethod of Birnboim and Doly (supra). All plasmids were cleaved with therestriction enzymes XhoI, BamHI and XbaI and the fragments were analyzedin 6% poly-acrylamide gels.

The restriction enzyme analysis indicated that the plasmids containedthe 3 desired fragments. Sequence analyses carried out as describedabove (Example 3, E) showed that adaptor 4 was fused correctly to theIFN-γ gene. These plasmids were designated pHis,His-Xa-IFN-γ (FIG. 21).The IFN-γ fusion protein (for amino acid sequence, see FIG. 41) codedfrom these plasmids was designated His,His-Xa-IFN-γ.

Example 7 Construction of Plasmid pHis,His-Ek-IFN-γ(-8) A. Principles

For the construction of the plasmid pHis,His-Ek-IFN-γ(-8), the following3 DNA fragments were linked with one another (FIG. 24): 1) a fragmentfrom plasmid pDS8/RBSII,SphI-His,His-Xa-BamHI containing the promoterP_(N25) x_(/O), the ribosomal binding site RBSII,SphI and the regioncoding for the adjacent histidines, which has been lengthened with theaid of adaptor 5 (FIGS. 11A-B) by a region coding for the recognitionsite of enterokinase (EK) (fragment 7, FIG. 22); 2) a fragment from theplasmid pIFN-γ(-8) which contains the gene for IFN-γ(-8) (fragment 8,FIG. 23): and 3) a fragment from the plasmid pDSS/RBSII,3A+5A having thereplication region and the β-lactamase gene (fragment 5, FIG. 19). Thepreparation of the last-named fragment has been described in Example 6.

B. Preparation of Fragment 7

4 pmol of plasmid pDS8/RBSII,SphI-His,His-Xa-BamHI were cleaved with therestriction enzyme NaeI. Subsequently, the enzyme was heat-inactivated,the sample was extracted with phenol, extracted with ether and the DNAwas precipitated as described. The sediment was taken up in 50 μl of TEbuffer. 1.5 pmol of the cleaved DNA were incubated in a volume of 200 μlwith 30 pmol of the phosphorylated adaptor 5 (FIGS. 11A-B) and 7U of T4DNA ligase for 14 hours in ligase buffer. After heat inactivation of theenzyme the DNA was cleaved with the restriction enzymes NdeI and XhoI.

Subsequently, the enzymes were heat-inactivated, the sample wasextracted with phenol, extracted with ether and the DNA was precipitatedas described above. The sediment was taken up in sample buffer, and themixture was incubated for 7 minutes at 65° C. Thereafter, the DNA wasseparated in a 6% polyacrylamide gel. 0.2 pmol of the XhoI/NdeI fragmentcontaining the promoter P_(N25) x_(/O), the ribosomal binding siteRBSII,SphI and the region coding for the neighbouring histidines and forthe recognition site of enterokinase, was isolated from the gel aspreviously described. This DNA fragment was designated fragment 7 (FIG.22).

C. Preparation of Fragment 8

0.5 pmol of the plasmid pIFN-γ(-8) was cleaved with the restrictionenzymes NdeI and XbaI. Subsequently, the enzyme was heat-inactivated,the sample was extracted with phenol, extracted with ether and the DNAwas precipitated as described. The sediment was taken up in samplebuffer and the mixture was incubated for 7 minutes at 65° C. The DNA wasthen separated in a 6% polyacrylamide gel. 0.05 pmol of the NdeI/XbaIfragment containing the IFN-γ(-8) gene, the terminator t_(o), the catgene and the terminator T1 was isolated from the gel as described. ThisDNA fragment was designated fragment 8 (FIG. 23).

D. Assembly of Plasmid pHis,His-Ek-IFN-γ(-8)

0.006 pmol of fragment 5 (FIG. 19). 0.02 pmol of fragment 7 (FIG. 22)and 0.005 pmol of fragment 8 (FIG. 23) were incubated in ligase bufferin a volume of 30 μl with 0.5 U of T4 DNA ligase for 3 hours at 150° C.After heat inactivation of the enzyme, the batch was transformed asdescribed above into E. coli strain M15 which contained the plasmidpDMI,l, and the transformation mixture was plated-out on LB plates whichcontained 100 μg/ml ampicillin, 25 μg/ml kanamycin and 5 μg/mlchloramphenicol.

After incubation of the plates for 24 hours at 37° C., 2 of thetransformants obtained were grown up in 10 ml of LB medium containing100 μg/ml ampicillin, 25 μg/ml kanamycin and 5 μg/ml chloramphenicol,and the plasmids were isolated according to the method of Birnboim andDoly (supra).

The plasmids were analyzed for size in 0.7% agarose gels and forcomposition with the aid of the restriction enzymes HindIII, HinfI,NdeI, SphI, XbaI and XhoI in 6% polyacrylamide gels. Both plasmidscontained the 3 desired DNA fragments in the correct orientation to oneanother. The sequence analyses, which were carried out as previouslydescribed (Example 3. E), indicated that the ribosomal binding siteRBSII,SphI with the subsequent elements had been linked correctly withthe IFN-γ(-8) gene. These plasmids were designated pHis,His-Ek-IFN-γ(-8)(FIG. 24). The IFN-γ fusion protein (amino acid sequence, FIG. 42) codedfrom these plasmids was designated His,His-Ek-TFN-γ(-8).

Example 8 Construction of Plasmid pHis,His-Xa-IFN-γ(-8)(Asn) A.Principles

For the construction of plasmid pHis,His-Xa-IFN-γ(-8)(Asn), thefollowing 3 DNA fragments were linked with one another (FIG. 26): 1) afragment from the plasmid pIFN-γ(-8) which contained the IFN-γ(-8) gene(fragment 8. FIG. 23, the preparation of which has been described inExample 7); 2) a fragment from the plasmidpDS8/RBSII,SphI-His,His-Xa-BamHI containing the promoter p_(N25) x_(/O),the ribosomal binding site RBSII,SphI and the region coding for theneighbouring histidines and for the recognition site of factor Xa, whichhas been lengthened with the aid of adaptor 6 (FIGS. 11A-B) so that, bylinkage with the fragment described under 1), the IFN-γ(-8) derivativeIFN-γ(-8)(Asn) is coded (fragment 9. FIG. 25); and 3) a fragment fromthe plasmid pDS5/RBSII, 3A+5A with the replication region and the genefor β-lactamase (fragment 5, FIG. 19, the preparation of which has beendescribed in Example 6).

B. Preparation of Fragment 9

2 pmol of plasmid pDS8/RBSII,SphI-His,His-Xa-BamHI were cleaved with therestriction enzyme NarI. Subsequently, the enzyme was heat-inactivated,the sample was extracted with phenol, extracted with ether and the DNAwas precipitated as described. The sediment was taken up in 50 μl of TEbuffer. 1 pmol of the cleaved DNA was incubated in a volume of 150 μlwith 30 pmol of phosphorylated adaptor 6 (FIGS. 11A-B) and 5U of T4 DNAligase for 14 hours at 20° C. in ligase buffer. After heat inactivationof the enzyme the DNA was cleaved with the restriction enzymes NdeI andXhoI.

Subsequently, the enzymes were heat-inactivated, the sample wasextracted with phenol, extracted with ether and the DNA was precipitatedas described. The sediment was is taken up in sample buffer and thebatch was incubated for 7 minutes at 65° C. The resulting DNA mixturewas then separated in a 6% poly-acrylamide gel. 0.25 pmol of the.XhoI/NdeI fragment with the promoter P_(N25) x_(/O), the ribosomalbinding site RBSII,SphI and the region which codes for the adjacenthistidines, for the recognition site of factor Xa and for the amino acidAsn was isolated from the gel as described. This DNA fragment wasdesignated fragment 9 (FIG. 25).

C. Assembly of Plasmid pHis,His-Xa-IFN-γ(-8)(Asn)

0.006 pmol of fragment 5 (FIG. 19). 0.005 pmol of fragment 8 (FIG. 23)and 0.02 pmol of fragment 9 (FIG. 25) were incubated in 30 μl of ligasebuffer and 0.5 U of T4 DNA ligase for 3 hours at 15° C. After heatinactivation of the enzyme, the batch was transformed as described aboveinto E. coli strain MIS which contained the plasmid pDMI,l, and thetransformation mixture was plated-out on LB plates containing 100 μg/mlampicillin and 25 μg/ml kanamycin. After incubation of the plates at 37°C. overnight 2 colonies were grown up as described in 10 ml of LB mediumcontaining 100 μg/ml ampicillin and 25 μg/ml kanamycin, and the plasmidswere isolated according to the method of Birnboim and Doly (supra).

The plasmids were analyzed with respect to their size in 0.7% agarosegels and with respect to their composition with the aid of therestriction enzymes HindIII, HinfI, NdeI, SphI, XbaI and XhoI in 6%polyacrylamide gels. One of the two plasmids contained the desired 3 DNAfragments in the correct orientation to one another.

Sequence analysis of this plasmid carried out as described above(Example 3. E) indicated that the ribosomal binding site RBSII,SphI withthe following elements had been correctly linked with the region whichcodes for IFN-γ(-8)(Asn). This plasmid was designatedpHis,His-Xa-IFN-γ(-8)(Asn) (FIG. 26). The IFN-γ fusion protein (foramino acid sequence, see FIG. 43) coded from this plasmid was designatedHis,His-Xa-IFN-γ(-8)(Asn).

Example 9 Construction of Plasmid p6xHis-DHFR A. Principles

For the construction of plasmid p6xHis-DHFR. the following DNA fragmentswere isolated and linked with each other (FIG. 28): 1) the signal unitof the plasmid pDS78/RBSII having the promoter N25OPSN25OP29 and theribosomal binding site RBSII, which has been linked with the adaptor 7(FIGS. 11A-B), (fragment 10, FIG. 27) and 2) the larger of the twoXhoI/BamHI fragments of the plasmid pDS78/RBSII (FIG. 28).

B. Preparation of Fragment 10

2 pmol of plasmid pDS78/RBSII were cleaved with the restriction enzymeBamHI. After heat inactivation of the enzyme, the sample was extractedwith phenol, extracted with ether and the DNA was precipitated asdescribed. The sediment was dissolved in 10 μl of ligase buffer. 50 pmolof the phosphorylated adaptor 7 (FIG. 27) dissolved in ligase bufferwere added to the cleaved plasmid, and the sample was incubated with 2 Uof ligase (22° C., hours).

After heat inactivation of the ligase, the sample was extracted withphenol, extracted with ether and the DNA was precipitated as described.The sediment was dissolved and the DNA was cleaved with the restrictionenzymes XhoI and BamHI. After adding sample buffer, heating the mixtureat 65° C. for 7 minutes and separating the DNA in a 6% polyacrylamidegel the XhoI/BamHI fragment having the promoter N25OPSN25OP29, theribosomal binding site RBSII and the region coding for 6 histidines wasisolated as described. This fragment was designated fragment 10 (FIG.27).

C. Preparation of the BamHI/XhoI Fragment of Plasmid pD78/RBSII

2 pmol of plasmid pDS78/RBSII were cleaved with the restriction enzymesXhoI and BamHI. After working-up the sample and gel electrophoresis, thefragment which contains the replication region (FIG. 28) was isolated.

D. Assembly of Plasmid p6xHis-DHFR

In each case 0.1 pmol of the specified fragments were incubated inligase buffer with 2 U of T4 DNA ligase (22° C., 3 hours). After heatinactivation of the enzyme the mixture was transformed as describedabove into E. coli strain M15 which contained the plasmid pDMI,l. Thetransformation mixture was plated-out on LB agar plates which contained100 μg/ml ampicillin and 25 μg/ml kanamycin, and the plates wereincubated at 370° C. overnight.

Individual colonies were grown up in 10 ml of LB medium as describedabove, and the plasmids were isolated according to the method ofBirnboim and Doly (supra). A restriction analysis with the enzymes XhoIand BamHI indicated that the plasmids contained the 2 desired fragments.Sequence analysis carried out as described above (Example 3. E)confirmed that adaptor 7 had been linked correctly with the ribosomalbinding site. These plasmids, which code for the DHFR fusion protein(His)₆ -mDHFR, were designated p6xHis-DHFR (FIG. 28).

EXAMPLE 10 Construction of Plasmid p4xHis-DHFR

The construction of the plasmid p4xHis-DHFR was carried out analogouslyto the construction of plasmid p6xHis-DHFR (Example 9). with thefollowing DNA fragments being isolated and linked with each other (FIG.30): 1) the signal unit of plasmid pDS78/RBSII having the promoterN25OPSN25OP29 and the ribosomal binding site RBSII, which has beenlinked with the adaptor 8 (FIGS. 11A-B), (fragment 11, FIG. 29) and 2)the larger of the two XhoI/BamHI fragments of plasmid pDS78/RBSII (FIG.30). The resulting plasmid p4xHis-DHFR codes for the DHFR fusion protein(His)₄ -mDHFR.

Example 11 Construction of Plasmid pRBSII-6xHis A. Principles

For the construction of plasmid pRBSII-6xHis, the following DNAfragments were isolated and linked with each other (FIG. 32): 1) theregion from plasmid pDS56/ RBSII having the terminator t_(o), the catgene and the terminator T1, which has been lengthened by the adaptor 9(which codes for 6 histidines), (fragment 12, FIG. 31) and 2) theXbaI/BamHI fragment from plasmid pDS56/RBSII having the replicationregion, the bla gene, the promoter N25OPSN25OP29 and the ribosomalbinding site RBSII (FIG. 32).

B. Preparation of Fragment 12

2 pmol of plasmid pDS56/RBSII were cleaved with the restriction enzymeHindIII. After working-up the sample, 50 pmol of phosphorylated adaptor9 were added to the cleaved plasmid, and the sample was incubated withT4 DNA ligase as previously described. After working-up the ligationbatch, the DNA was cleaved with the restriction enzymes BamHI and XbaI,and the BamHI/XbaI fragment having the region coding for 6 histidines,the terminator t_(o), the cat gene and the terminator T1 was isolated asdescribed. This fragment was designated fragment 12 (FIG. 31).

C. Preparation of the XbaI/BamHI Fragment of Plasmid pDS56/RBSII

2 pmol of plasmid pDS56/RBSII were cleaved with the restriction enzymesXbaI and BamHI, and the fragment having the replication region, the blagene, the promoter N25OPSN25OP29 and the ribosomal binding site RBSIIwas isolated as described (FIG. 32).

D. Assembly of Plasmid pRBSII-6xHis

In each case 0.1 pmol of the isolated fragments were, as described(Example 9,D), ligated and subsequently transformed into the E. colistrain M15 (pDMI,1). After plating and incubation (Example 9, D),individual colonies were grown up in 10 ml of medium as described andthe plasmids were isolated according to the method of Birnboim and Doly(supra). A restriction analysis with the enzymes BamHI and XbaIindicated that the plasmids contained the 2 desired fragments. Thesequence analysis (Example 3, E) confirmed that adaptor 9 had beenintroduced correctly, into the plasmid DNA. These plasmids weredesignated pRBSII-6xHis.

Example 12 Construction of Plasmid pRBSII-4xHis

The construction of plasmid pRBSII-4xHis was carried out analogously tothe construction of plasmid pRBSII-6xHis (Example 11). with thefollowing DNA fragments being isolated and linked with each other (FIG.34): 1) the region from the plasmid pDS56/RBSII having the terminatort_(o), the cat gene and the terminator T1, which has been lengthened byadaptor 10 (which codes for 4 histidines), (fragment 13. FIG. 33) and 2)the XbaI/BamI fragment from plasmid pDS56/RBSII having the replicationregion, the bla gene, the promoter N25OPSN25OP29 and the ribosomalbinding site RBSII (FIG. 34).

Example 13 Construction of Plasmid pRBSII-2xHis

The construction of plasmid pRBSII-2xHis was carried out analogously tothe construction of plasmid pRBSII-6xHis (Example 11), with thefollowing DNA fragments being isolated and linked with each other (FIG.36): 1) the region from plasmid pDS56/RBSII having the terminator t_(o),the cat gene and the terminator T1, which has been lengthened by theadaptor 11 (which codes for 2 histidines), (fragment 14, FIG. 35) and 2)the XbaI/BamHI fragment from the plasmid pDS56/RBSII having thereplication region, the bla gene, the promoter N25OPSN25OP29 and theribosomal binding site RBSII (FIG. 36).

Example 14 Construction of Plasmid pDHFR-6xHis A. Principles

For the construction of plasmids pDHFR-6xHis, the following DNAfragments were isolated and linked with each other (FIG. 37): 1) theXbaI/BglII fragment from plasmid pDS78/RBSII having the replicationregion, the bla gene, the promoter N25OPSN25OP29, the ribosomal bindingsite RBSII and the dhfr gene and 2) the BglII/XbaI fragment from plasmidpRBSII-6xHis having the region coding for 6 histidines, the terminatort_(o), the cat gene and the terminator T1. The resulting plasmidpDHFR-6xHis codes for the DHFR fusion protein Met-mDHFR-(His)₆.

B. Preparation of the XbaI/BqlII Fragment of Plasmid pDS78/RBSII

2 pmol of plasmid pDS78/RBSII were cleaved with the restriction enzymesXbaI and BglII. After working-up the sample the XbaI/BglII fragmenthaving the replication region, the bla gene, the promoter N25OPSN25OP29,the ribosomal binding site RBSII and the dhfr gene was isolated asdescribed.

C. Preparation of the BqlII/XbaI Fragment of Plasmid pRBSII-6xHis

2 pmol of plasmid pRBSII-6xHis were cleaved with the restriction enzymesBglII and XbaI. After working-up the sample the BglII/XbaI fragmenthaving the region coding for 6 histidines, the terminator t_(o), the catgene and the terminator T1 was isolated as described.

D. Assembly of Plasmid pDHFR-6xHis

In each case 0.1 pmol of the isolated fragments were, as described(Example 9,D), ligated and transformed into E. coli strain M15 (pDMI,1).After plating and incubation (Example 9, D) individual colonies weregrown up in 10 ml of medium as described and the plasmids were isolatedaccording to the method of Birnboim and Doly (supra). A restrictionanalysis with the enzymes XbaI and BglII indicated that the twofragments had been linked with one another in the desired manner. Theseplasmids were designated pDHFR-6xHis.

Example 15 Construction of Plasmid pDHFR-2xHis

The construction of plasmid pDHFR-2xHis, which codes for the DHFR fusionprotein Met-mDHFR-(His)₂, was carried out analogously to theconstruction of plasmid pDHFR-6xHis (Example 14), with the following DNAfragments being isolated and linked with each other (FIG. 38): 1) theXbaI/BglII fragment from plasmid pDS78/RBSII having the replicationregion, the bla gene, the promoter N25OPSN25OP29, the ribosomal bindingsite RBSII and the dhfr gene and 2) the BglII/XbaI fragment from plasmidpRBSII-2xHis having the region coding for 2 histidines, is theterminator t_(o), the cat gene and the terminator T1.

Example 16 Construction of Plasmid p4xHis-DHFR-4xHis

The construction of plasmid p4xHis-DHFR-4xHis, which codes for the DHFRfusion protein (His) 4- mDHFR-(His)₄, was carried out analogously to theconstruction of plasmid pDHFR-6xHis (Example 14), with the following DNAfragments being isolated and linked with each other (FIG. 39): 1) TheXbaI/BglII fragment from plasmid p4xHis-DHFR having the replicationregion, the bla gene, the promoter N25OPSN25OP29, the ribosomal bindingsite RBSII,4xHis and the dhfr gene and 2) the BglII/XbaI fragment fromplasmid pRBSII-4xHis having the region coding for 4 histidines, theterminator t_(o), the cat gene and the terminator T1.

Example 17 Preparation of the NTA Resin

41.7 g of bromoacetic acid were dissolved in 150 ml of 2 N sodiumhydroxide solution and cooled to 0°. Thereto there was slowly addeddropwise at 0° C. while stirring a solution of 42 g of N.sup.ε-Z-L-lysine in 225 ml of 2N sodium hydroxide solution. After 2 hours,the cooling was o removed and the mixture was stirred overnight. Thereaction mixture was then held at 50° C. for 2 hours, and 450 ml of 1Nhydrochloric acid were subsequently added. After the mixture had beencooled, the separated crystals were filtered off. The product wasdissolved in 1N sodium hydroxide solution and again precipitated withthe same amount of 1N hydrochloric acid and filtered off. There wereobtained 40 g ofN-[5-benzyloxycarbonylamino-1-carboxypentyl]-iminodiacetic acid in theform of white crystals, m.p. 172°-174° C. (dec.), [CL]_(D) + 9.90° (c =1; 0.1N NAOH).

7.9 g of the lysine derivative obtained were dissolved in 49 ml of 1Nsodium hydroxide solution and, after the addition of a spatula tip of 5%palladium on charcoal (Pd/C). hydrogenated at room temperature andnormal pressure. The catalyst was filtered off and the filtrate wasevaporated. There resulted 6.2 g ofN-[5-amino-1-carboxypentyl]-iminodiacetic acid whose structure, NH₂(CH₂)₄ --CH(COOH)--N--(CH₂ COOH)₂, was confirmed by the NMR spectrum.

100 ml of Sepharose® (D CL-6B (Pharmacia) were washed twice on a glasssuction filter with about 600 ml of water and then reacted at 30° C. for4 hours in a 500 ml round flask with 16 ml of 4N sodium hydroxidesolution and 8.22 ml of epibromohydrin. The total volume of the reactionmixture was 200 ml. The activated Sepharose was subsequently filteredoff, washed neutral with water and transferred back into the reactionvessel.

6.5 g of N-[5-amino-1-carboxypentyl)-iminodiacetic acid were dissolvedin 50 ml of water and added to the activated Sepharose together with10.6 g of solid sodium carbonate. The mixture was stirred slowly at 600°C. overnight. The resulting chelate resin with the formula[Sepharose®CL-6B]--O--CH₂ --CH(OH)--CH₂ --NH--(CH₂)₄ --CH(COOH)--N--(CH₂COOH)₂ (NTA resin) was subsequently washed in a chromatography column insuccession with 500 ml of water, 100 ml of aqueous NISO₄.6H₂ O (2 wt.%), 200 ml of water, 200 ml of 0.2M acetic acid (containing 0.2M NaCland 0.1 wt./vol. % Tween 20) and 200 ml of water. The nickel ionconcentration in the resulting chelate resin of the formula[Sepharose®CL-6B]--O--CH₂ --CH(OH)--CH₂ --NH-----(CH₂)₄--CH(COOH)--N(CH₂ COO⁻)₂ Ni²⁺ amounted to about 7.1 micromol/ml.

Example 18 Metal Chelate Affinity Chromatography With Purified IFN-γ

A column (diameter, 1.6 cm; length, 7.0 cm) was filled with metal-freechelate resin of the formula [Sepharose® CL-6B]--O--CH₂ --CH(OH)--CH₂--NH--(CH₂)₄ -- CH(COOH)--N(CH₂ COOH)₂ (NTA resin) and the resin wasbrought into the nickel form by rinsing with a three-fold column volumeof 0.1M NiSO₄.5H₂ O and subsequently washing with a three-fold columnvolume of 0.2M acetic acid. The resin was subsequently equilibrated with0.1M Tris.HCl buffer (pH 7.5) and 0.5M NaCl (throughflow in each case 60ml/hr.).

1 mg of purified IFN-γ (Example 3, amino acid sequence see FIGS. 40A-B)was taken up in 3 ml of equilibration buffer and applied to the column.By means of enzyme immunoassay [Gallati, H., J. Clin. Chem. Clin.Biochem. 20, 907-914 (1982)] it could be detected that in spite of thetwo internal protein structural elements Gly-His-Ser and Ile-His-Glu nobinding to the NTA column took place.

Example 19 Purification of His,His-Xa-IFN-γ By Means of NTA Resin

E. coli M15 cells containing plasmids pDMI.l and pHis,His-Xa-IFN-γ(Example 6) were left to grow in 1 liter of LB medium containing 100μg/ml ampicillin and 25 μg/ml kanamycin at 370° C. up to an opticaldensity of OD₆₀₀ = 0.6. Then, IPTG was added (final concentration 0.5mM) and the cells were incubated for a further 4 hours. Subsequently,the cells were separated (5 g wet weight) from the culture medium bycentrifugation (4000 × g, 10 min. 40° C.) and extracted with 15 ml of 7Mguanidine.HCl and 0.01M sodium borate (pH 8) (1 hour, 4° C., magneticstirrer). The thus-obtained crude extract was centrifuged (10,000 × g,151, 4° C.). the supernatant was diluted 10-fold with 0.1M Tris.HClbuffer (pH 7.5) and 0.5M NaCl, again centrifuged (10,000 × g, 15minutes, 4° C.) and pumped up on the same NTA column as described inExample 18.

The column was subsequently washed with equilibrating buffer until theUV detector (280 nm) again showed the base value. The elution of theHis,His-Xa-IFN-γ was effected by lowering the pH value to 5.5. By meansof enzyme immunoassay [Gallati, H., supra] it could be detected thatthis protein was quantitatively absorbed on the NTA column and waseluted only by lowering the pH value. By means of SDS-polyacrylamide gelelectrophoresis and RP-18 HPLC it could be detected that the proteinobtained was pure His,His-Xa-IFN-γ (purity >90%). The expectedamino-terminal sequence Met-His-His-Ala-Gly-Ile-Glu-Gly-Arg-Gln . . .was confirmed by Edman degradation.

Example 20 Purification of His,His-Ek-IFN-γ(-8) By Means of NTA Resin

His,His-Ek-IFN-γ(-8) (Example 7) was expressed in E. coli in ananalogous manner to Example 19, extracted and purified via the NTAcolumn. This fusion protein was also bonded to the NTA column at pH 7.5and was eluted in pure form (purity > 90%) by lowering the pH value to5.5. The expected sequence Met-His-His-Ala-Gly-Asp-Asp-Asp-Asp-Lys-Gln .. . was confirmed by Edman degradation.

Example 21 Purification of His,His-Xa-IFN-γ(-8)(Asn) By Means of NTAResin

His,His-Xa-IFN-γ(-8)(Asn) (Example 8) was expressed in E. coli in ananalogous manner to Example 19, extracted and purified via the NTAcolumn. This protein was also bonded to the NTA column at pH 7.5 and waseluted in pure form (purity > 90%) by lowering the pH value to 5.5.

1 mg of the thus-obtained His-His-Xa-IFN-γ(-8)(Asn) was dialyzed against0.1M Tris.HCl (pH 7.5), 0.5M NaCl and 1 mM CaCl₂. The dialysate (5 ml)was treated with 100 μl (lU) of coagulation factor Xa (BoehringerMannheim) and incubated for 16 hours at 220° C. The enzymaticdegradation of the His-His-affinity peptide was determined by means ofSDS-polyacrylamide gel electrophoreses.

For the purpose of separating salts, bovine serum albumin (constituentof the commercial factor Xa preparation) and factor Xa, the incubationmixture was firstly dialyzed against water, then lyophilized andsubsequently chromatographed on a RP-18 HPLC column (Nucleosil 5Cl8column from Brownlee Labs, running agent 0.1% trifluoroacetic acid,gradient with acetonitrile, throughflow 1 ml/min). The resultingpurified protein was then freed is from solvent and subjected to anEdman degradation. The expected amino-terminal sequence Gln-Asn-Pro-Tyr. . . could be confirmed by means of this method.

This experiment shows that the affinity sequence at the NH₂ terminus ofthe His-His-Xa-IFN-γ(-8)(Asn) can be cleaved off cleanly after the metalchelate affinity chromatography.

Example 22 Purification of (His)₆ -mDHFR By Means of NTA Resin in 6 MGuanidine.HCl

(His)₆ -mDHFR (Example 9) was expressed in E. coli in an analogousmanner to Example 19. The cells were extracted with 6M guanidine.HCl in0.1M sodium phosphate buffer (pH 8.0) (5 ml of buffer solution per 1 gof cells, 1 hr., 22° C., magnetic stirrer). The thus-produced crudeextract was subsequently centrifuged and the supernatant was pumped onto the same NTA column as described in Example 18. The chromatographywas carried out analogously to Example 19 with the exception of thebuffer solutions used. The buffers used contained in each case 6Mguanidine.HCl in 0.1M sodium phosphate buffer having the following pHvalues: pH 8.0 to apply the proteins, pH 6.0 to wash-out the non-boundE. coli proteins and pH 4.5 in order to elute the (His)₆ -mDHFR. Theeluate obtained was dialyzed against water and subsequently lyophilized.By means of SDS-polyacrylamide gel electrophoresis, it was shown thatthe protein obtained was pure (His)₆ -mDHFR (purity >90%). The expectedsequence Met-Arg-Gly-Ser-His-His-His-His-His-His--Gly-Ser-Ile-Met . . .was confirmed by Edman degradation.

Example 23 Purification of (His)₄ -mDHFR-(His)₄ By Means of NTA Resin in6M Guanidine.HCl

(His)₄ -mDHFR-(His)₄ (Example 16) was expressed in E. coli in ananalogous manner to Example 19, extracted and purified via the NTAcolumn. In place of the stepwise gradients used in Example 22 there wasused for the elution a linear pH gradient (pH 8.0 to pH 4.0. 2 hours).(His) 4- mDHFR-(His)4fusion protein was eluted at pH 4.9 and had apurity of at least 90%.

Example 24 Purification of Met-mDHFR-(His) By Means of NTA Resin in 6MUrea

Met-mDHFR-(His) (Example 14) was expressed in E. coli in an analogousmanner to Example 19. The centrifuged-off cells were extracted with 6Murea in 0.05M sodium phosphate buffer (pH 7.5) (1 g of cells per 10 mlof buffer solution) and ultrasound (10 minutes). After centrifugation ofthe cell debris, the supernatant was applied to a NTA column (4.5 cm ×2.6 cm) equilibrated with extraction buffer. After washing the columnwith extraction buffer the Met-mDHFR-(His)₆ fusion protein was elutedwith a linear PH gradient of PH 7.5 (extraction buffer) to PH 4.8 (0.05Msodium phosphate buffer containing 6M urea) during 5 hours and at a pumpvelocity of 18 ml per hour. The fractions which contained protein wereanalyzed by means of SDS-polyacrylamide gel electrophoresis. 9 mg ofMet-mDHFR-(His)₆ fusion protein with a purity of >90% were obtained.

Example 25 Purification of Met-mDHFR-(His)₂ By Means of NTA Resin

Met-mDHFR-(His)₂ (Example 15) was expressed in E. coli in an analogousmanner to Example 19. The centrifuged-off cells in 0.05M potassiumphosphate buffer (pH 8.0), containing 0.1M potassium chloride and 0.1%Tween 20, were treated with ultrasound in an ice bath during 15 minutes(1 g of cells per 10 ml of buffer solution). Subsequently, the celldebris were centrifuged off and the clear supernatant was placed on aNTA column (4.6 cm × 2.6 cm) equilibrated with extraction buffer. Thecolumn was washed with extraction buffer and the Met-mDHFR-(His)₂ fusionprotein was eluted with a linear pH gradient of pH 8.0 (extractionbuffer) to pH 5.0 (0.05M potassium phosphate buffer containing 0.1Mpotassium chloride and 0.1% Tween 20) during 10 hours and at a pumpvelocity of 50 ml per hour. The peak fractions of the eluent wereanalyzed by means of SDS-polyacrylamide gel electrophoresis. 7 mg ofMet-mDHFR-(His).sub. 2 fusion protein with a purity of >85% wereobtained.

3 mg of the thus-obtained fusion protein Met-mDHFR-(His)₂ were dialyzedagainst 0.05M Tris.HCl (pH 6.0) at 60° C. The protein solution was thenadjusted to pH 9.0 with 0.5M NAOH and incubated at 37° C. in thepresence of 8.5 units of carboxypeptidase A from bovine pancreas (Serva,Feinbiochemica, Heidelberg BRD). After 0, 15, 30, 90 and 180 minutes,samples were removed and analyzed for their histidine content by meansof HPLC. After 480 minutes the pH value was lowered to 8.0 and thereaction mixture was pumped onto an NTA column equilibrated with 0.05Mpotassium phosphate buffer (pH 8). The protein contained in the reactionmixture was detected in the flow-through by means of SDS-polyacrylamidegel electrophoresis. In addition, an amount of histidine residuesincreasing with time was detected in the samples which had been removedfrom the protein solution after 15, 30, 90 and 180 minutes.

This experiment shows that the affinity sequence at the carboxylterminus can be removed cleanly after the purification on NTA resin.

What is claimed is:
 1. A DNA sequence coding for a fusion proteincomprising a biologically active polypeptide or protein linked by itsamino- or carboxy-terminus to one or two affinity peptides, whichpeptides have from 2 through 6 adjacent histidine residues:
 2. The DNAsequence of claim 1 in which the affinity peptides of the fusion proteinhave the formula:

    R.sup.1 --(His).sub.2-4 --R.sup.2,

where R¹ is hydrogen or from 1 to about 30 amino acids; and R² is Q,Q-Ile-Glu-Gly-Arg- or Q-Asp-Asp-Asp-Asp-Lys-, where Q is a peptide bondor from 1 to about 30 amino acids.
 3. A recombinant vector comprising avector and a DNA sequence coding for a fusion protein comprising abiologically active polypeptide or protein linked by its amino- orcarboxyl-terminus to one or two affinity peptides, which peptides havefrom two through six adjacent histidine residues, wherein therecombinant vector is capable of directing expression of the DNAsequence in a compatible unicellular host organism.
 4. The recombinantvector of claim 3 in which the affinity peptides of the fusion proteinhave the formula:

    R.sup.1 --(His).sub.2-4 --R.sup.2,

where R¹ is hydrogen or from 1 to about 30 amino acids; and R2 is Q,Q-Ile-Glu-Gly-Arg- or Q-Asp-Asp-Asp-Asp-Lys-, where Q is a peptide bondor from 1 to about 30 amino acids.
 5. A unicellular organism containinga recombinant vector comprising a vector and a DNA sequence coding for afusion protein comprising a biologically active polypeptide or proteinlinked by its amino- or carboxyl-terminus to one or two affinitypeptides, which peptides have from two through six adjacent histidineresidues, wherein the unicellular organism is capable of expressing theDNA sequence.
 6. The unicellular organism of claim 5 which is E. colistrain M15.
 7. A process for producing a fusion protein comprising:(a)culturing a unicellular organism containing a recombinant vectorcomprising a vector and a DNA sequence coding for a fusion proteincomprising a biologically active polypeptide or protein linked by itsamino- or carboxyl-terminus to one or two affinity peptides, whichpeptides have from two through six adjacent histidine residues, whereinthe unicellular organism is capable of expressing the DNA sequence,under conditions in which the fusion protein is produced; and (b)isolating the fusion protein from the culture.
 8. The process of claim 7in which the fusion protein is isolated by a proc:: comprising:(a)contacting a lysate or extract of the unicellular organism containingthe fusion is protein with a metal chelate resin of the structure:

    carrier-spacer--NH--(CH.sub.2).sub.X --CH(CO.sub.2 H)--

where X is an integer from 2 to 4,under conditions in which the fusionprotein binds to the resin to produce a resin-fusion protein complex;(b) washing the resin-fusion protein complex with a buffer to removeunbound proteins and other materials; and (c) eluting the bound fusionprotein from the washed resin-fusion protein complex.
 9. The process ofclaim 8 in which at least one of the links between the biologicallyactive polypeptide or protein and the affinity peptides of the isolatedfusion protein can be cleaved with a protease, and such links arecleaved after isolation by such protease.
 10. The process of claim 8 inwhich the carrier is a beaded agarose.
 11. The process of claim 8 inwhich the spacer is --O--CO-- or --O--CH₂ --CH(OH)--CH₂ --.